During FY12, we focussed mainly on the following four subprojects: (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (cAg, capsids) and e-antigen (eAg, unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into shells of two different sizes, we obtained, in 1997, a cryo-EM density map in which we visualized the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. We went on to investigate the antigenic diversity of HBV by using cryo-electron microscopy (EM) to characterize the conformational epitopes of seven monoclonal antibodies by cryo-EM and molecular modeling. In FY12, we completed and published two projects mentioned in last year's report. In one (2), we sought to relate our previous work with murine monoclonals to the immunological response of infected humans by investigating the binding of polyclonal anti-cAg antibodies from a patient. The observed Fab-related density could be reproduced by modeling with just five Fabs whose locations match those of the murine monoclonals previously studied. These results validate the mouse as a model system. We also investigated the reactivity of an antibody found in copious quantities in the livers of patients suffering from fulminant hepatitis and found that this antibody binds tangentially to a novel site on the side of the spike. These results support the idea that antibodies with particular specificities may correlate with different stages of disease progression. We also investigated the properties of eAg), which differs from cAg in having at its N-terminus an additional ten residues, a remnant of its propeptide. eAg and cAg are antigenically distinct but are cross-reactive (1). One eAg-specific Fab forms a stable complex with eAg that yielded crystals that diffract to 0.33 nm resolution. In the resulting structure, the eAg monomer is seen to have a similar fold to the cAg monomer but an entirely different mode of dimerization, related by a rotation of 140 degrees. This switch accounts for the profound differences in assembly properties and antigenicity between the two proteins. These results have been submitted for publication. (2) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system. The tailed phages afford an excellent model for herpesvirus capsids, reflecting common evolutionary origins. In FY12, we pursued the following investigations. (2a) Regulation of genome packaging. The capsids of double-stranded RNA viruses serve as compartments for the replication and transcription of the viral genomes. We investigate the structural basis of this remarkable phenomenon in the phage phi6 system, which has a tripartite genome. In FY08, we reported the location of the P2 polymerase inside the viral procapsid. In FY10, we completed a study using cryo-electron tomography to map the distributions of P2-occupied sites and of the external sites occupied by P4, the packaging ATPase. In FY11, we investigated the expansion transformation undergone by the procapsid during RNA packaging. To investigate procapsid transformation, we induced expansion in vitro by acidification, heating, and elevated salt concentration. The results identify two structural intermediates between the procapsid and the mature capsid (7). Phi6 has an additional protein component, P7, which functions as a packaging accessory protein with involvement also in assembly. However, its location has been unknown. In FY12, we used cryo-EM to localize P7 by difference mapping between procapsids with different compositions (8). We found that P7 resides on the interior surface of the procapsid at sites that overlap those of P2, indicating competition between these two proteins and implying that substoichiometric quantities of both are sufficient to fulfil their biological functions. The P7 binding sites are arranged around the three-fold axes, suggesting that the protein promotes assembly by stabilizing an initiation complex. (2b) PhiKZ is a large and complex virus that infects the pathogenic bacterium Pseudomonas aeruginosa. The virion has a large icosahedral capsid containing densely packed DNA (280kbp) as well as a proteinaceous inner body which is invisible in cryo-electron micrographs because of contrast-matching with the surrounding DNA. We found, serendipitously, that the inner body is exceptionally sensitive to electron irradiation and explodes into bubbles of gaseous products at doses that leave the surrounding capsid only slightly blurred. We developed a computational method of analyzing these bubblegrams to locate the inner body in individual nucleocapsids and then to determine its structure (5). The inner body is 24 nm wide, 105 nm long, and consists of multiple stacked tiers with 6-fold symmetry. Mass spectrometry and SDS-PAGE indicate that the inner body has five major proteins, present in 100-200 copies each, as well as a number of minor proteins (6). The shape and position of the inner body suggest that it plays a role of organizer in the DNA packaging process. A working hypothesis is that inner body proteins are injected into the host cell along with the DNA where they fulfil functions needed early in infection. (3) Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope (3). The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The HSV-1 tegument is known to contact the capsid at its vertices and two proteins, UL36 and UL37, had been identified as candidates for this interaction. We showed by cryo-EM that capsids with and without UL37 exhibit a vertex-associated density that represents the ordered portion of UL36 (336 kDa).These observations (4) support the hypothesis that UL36 provides a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding of UL36 and UL37. (4) While much of the research described above was performed with icoshedral capsids which may be imaged by high-resolution image reconstruction, many viruses do not conform to this symmetry. Nevertheless, their three-dimensional structures may be studied by cryo-electron tomography. We published the first such analysis of a pleiomorphic virus - herpes simplex virus type 1 - in 2003 and have gone on to make numerous other applications. In addition to completing studies of Rubella virus (13), Newcastle disease virus (12), immature adenovirus (15), and maturing Dengue virus (14), in FY12 we studied how influenza virus responds to acidic pH (9). Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. At pH 4.9, we observed dramatic changes in morphology: elongated particles were no longer observed; larger particles representing fused virions appeared; the HA spikes became conspicuously disorganized; a layer of M1 matrix protein was no longer resolved on most virions; and the ribonucleoprotein complexes (RNPs) coagulated on the interior surface of the virion. These observations have illuminated the cell entry pathway of influenza virus.